Molecular oxygen is utilized by aerobic organisms to perform a variety of demanding oxidative transformations, such as the conversion of cysteine to cysteine sulfinic acid catalyzed by cysteine dioxygenase (CDO). However, aerobic metabolism also leads to various side reactions that produce reactive oxygen species, such as the superoxide anion (O2-). Nature has therefore developed an effective strategy for O2- removal that involves metalloenzymes known as superoxide dismutases (SODs), which require either Fe, Mn, Cu/Zn, or Ni metal cofactors for catalytic activity. Found in all aerobic organisms, SODs disproportionate the superoxide radical to O2 and H2O2. The Long-Term Objectives of the research outlined in this proposal are: 7 To identify key geometric and electronic structural features of the Fe- and MnSODs that contribute to the high catalytic rates of these enzymes. 7 To obtain molecular level insight into the reaction mechanisms of SODs and CDO. 7 To utilize our knowledge for engineering novel enzymatic functions into FeSOD. With these goals in mind, we have formulated the following Specific Aims: 1. Elucidate the mechanism of long-range tuning of the active site properties in Fe- and MnSODs. 2. Explore the means by which the so-called cambialistic SODs overcome the challenge of providing an active-site environment that tolerates both Fe- and Mn-supported activity. 3. Obtain molecular-level insight into the catalytic mechanisms of Fe- and MnSODs. 4. Identify key structural elements for Ni-supported SOD activity. 5. Investigate structure/function relationships and the catalytic mechanism of CDO. 6. Engineer CDO activity into FeSOD. To accomplish these goals, we will use a combined spectroscopic/computational approach for studying the resting states and substrate (analog) complexes of the native enzymes and selected mutants. The Fe- and MnSODs provide almost ideal protein scaffolds for investigating the mechanisms of long- range tuning of active-site properties, such as the metal ion reduction potential and substrate (analog)/active site interactions. By extending our studies to NiSOD and CDO, we can test and refine our hypotheses regarding the principles by which outer-sphere amino acid residues contribute to the optimization of metalloenzyme active sites. From a practical point of view, insights gained in our proposed studies may provide the basis for the rational design of SOD and CDO mimics for pharmaceutical applications, such as the treatment of Alzheimer disease and Parkinson disease. Both the superoxide radical anion and free cysteine have been shown to play a role in several neurodegenerative diseases, including motor neuron disease, Parkinson disease, and Alzheimer disease. Under normal circumstances, the concentration of these species is maintained at very low levels by superoxide dismutases and cysteine dioxygenase, which are the focus of this proposal. Insights gained in our proposed studies may provide a suitable basis for the rational design of enzyme mimics for pharmaceutical applications, such as the treatment of neurodegenerative diseases.